Mitochondrial RNA modifications shape metabolic plasticity in metastasis.

Aggressive and metastatic cancers show enhanced metabolic plasticity1, but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) (refs.2-4)-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m5C at position 34 in mitochondrial tRNAMet. m5C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m5C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m5C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis.

Molecular basis for the faithful replication of 5-methylcytosine and its oxidized forms by DNA polymerase ß.

DNA methylation is an epigenetic mark that regulates gene expression in mammals. One method of methylation removal is through ten-eleven translocation-catalyzed oxidation and the base excision repair pathway. The iterative oxidation of 5-methylcytosine catalyzed by ten-eleven translocation enzymes produces three oxidized forms of cytosine: 5-hydroxmethylcytosine, 5-formylcytosine, and 5-carboxycytosine. The effect these modifications have on the efficiency and fidelity of the base excision repair pathway during the repair of opposing base damage, and in particular DNA polymerization, remains to be elucidated. Using kinetic assays, we show that the catalytic efficiency for the incorporation of dGTP catalyzed by human DNA polymerase ß is not affected when 5-methylcytosine, 5-hydroxmethylcytosine, and 5-formylcytosine are in the DNA template. In contrast, the catalytic efficiency of dGTP insertion decreases ∼20-fold when 5-carboxycytosine is in the templating position, as compared with unmodified cytosine. However, DNA polymerase fidelity is unaltered when these modifications are in the templating position. Structural analysis reveals that the methyl, hydroxymethyl, and formyl modifications are easily accommodated within the polymerase active site. However, to accommodate the carboxyl modification, the phosphate backbone on the templating nucleotide shifts ∼2.5 Å to avoid a potential steric/repulsive clash. This altered conformation is stabilized by lysine 280, which makes a direct interaction with the carboxyl modification and the phosphate backbone of the templating strand. This work provides the molecular basis for the accommodation of epigenetic base modifications in a polymerase active site and suggests that these modifications are not mutagenically copied during base excision repair.

Diagnostic utility of dual 5-hydroxymethylcytosine/Melan-A immunohistochemistry in differentiating nodal nevus from metastatic melanoma: An effective first-line test for the workup of sentinel lymph node specimen.

BACKGROUND: Distinguishing benign nodal nevus from metastatic melanoma can be diagnostically challenging, with important clinical consequences. Recently, the loss of epigenetic marker, 5-hydroxymethylcytosine (5-hmC) expression by immunohistochemistry has been found in melanomas and atypical melanocytic neoplasms. METHODS: About 41 metastatic melanomas and 20 nodal nevi were retrieved. Nuclear 5-hmC (brown) and cytoplasmic Melan-A Red (red) double immunohistochemical staining was performed. RESULTS: Total or partial loss of nuclear expression of 5-hmC was noted in 40/41 metastatic melanomas; these tumor cells were strongly positive for Melan-A Red, except in one case of desmoplastic melanoma. All cases of nodal nevus showed uniformly retained nuclear expression of 5-hmC accompanied by strong Melan-A Red cytoplasmic staining. In two cases containing both nodal nevus and metastatic melanoma, all tumor cells were positive for Melan-A Red, but a nuclear expression of 5-hmC was selectively absent only in the melanoma tumor cells. CONCLUSION: Dual 5-hmC/Melan-A Red immunohistochemistry is highly specific in distinguishing nodal nevus from metastatic melanoma. Our protocol for brown and red chromogens used in this study provides excellent color contrast and is easy to interpret. Furthermore, this dual staining method allows the preservation of limited tumor tissue, which could be used for potential molecular studies.

Chronic ethanol-mediated hepatocyte apoptosis links to decreased TET1 and 5-hydroxymethylcytosine formation.

The 5-hydroxymethylcytosine (5hmc) is a newly identified epigenetic modification thought to be regulated by the TET family of proteins. Little information is available about how ethanol consumption may modulate 5hmC formation and alcoholic liver disease (ALD) progression. A rat ALD model was used to study 5hmC in relationship to hepatocyte apoptosis. Human ALD liver samples were also used to validate these findings. It was found that chronic ethanol feeding significantly reduced 5hmC formation in a rat ALD model. There were no significant changes in TET2 and TET3 between the control- and ethanol-fed animals. In contrast, methylcytosine dioxygenase TET1 (TET1) expression was substantially reduced in the ethanol-fed rats and was accompanied by increased hepatocyte apoptosis. Similarly, knockdown of TET1 in human hepatocyte-like cells also significantly promoted apoptosis. Down-regulation of TET1 resulted in elevated expression of the DNA damage marker, suggesting a role for 5hmc in hepatocyte DNA damage as well. Mechanistic studies revealed that inhibition of TET1 promoted apoptotic gene expression. Similarly, targeting TET1 activity by removing cosubstrate promoted apoptosis and DNA damage. Furthermore, treatment with 5-azacitidine significantly mimics these effects, suggesting that chronic ethanol consumption promotes hepatocyte apoptosis and DNA damage by diminishing TET1-mediated 5hmC formation and DNA methylation. In summary, the current study provides a novel molecular insight that TET1-mediated 5hmC is involved in hepatocyte apoptosis in ALD progression.-Ji, C., Nagaoka, K., Zou, J., Casulli, S., Lu, S., Cao, K. Y., Zhang, H., Iwagami, Y., Carlson, R. I., Brooks, K., Lawrence, J., Mueller, W., Wands, J. R., Huang, C.-K. Chronic ethanol-mediated hepatocyte apoptosis links to decreased TET1 and 5-hydroxymethylcytosine formation.

PML Recruits TET2 to Regulate DNA Modification and Cell Proliferation in Response to Chemotherapeutic Agent.

Aberrant DNA methylation plays a critical role in the development and progression of cancer. Failure to demethylate and to consequently reactivate methylation-silenced genes in cancer contributes to chemotherapeutic resistance, yet the regulatory mechanisms of DNA demethylation in response to chemotherapeutic agents remain unclear. Here, we show that promyelocytic leukemia (PML) recruits ten-eleven translocation dioxygenase 2 (TET2) to regulate DNA modification and cell proliferation in response to chemotherapeutic agents. TET2 was required by multiple chemotherapeutic agents (such as doxorubicin) to prmote 5-hydroxymethylcytosine (5hmC) formation. Stable isotope labeling with amino acids in cell culture, followed by immunoprecipitation-mass spectrometry, identified potential binding partners of TET2, of which PML mostly enhanced 5hmC formation. PML physically bound to TET2 via the PML C-terminal domain and recruited TET2 to PML-positive nuclear bodies. This interaction was disrupted by the PML-RARA t(15;17) mutation, which stems from chromosomal translocation between DNA encoding the C-terminal domain of PML and the retinoic acid receptor alpha (RARA) gene. In response to chemotherapeutic drugs, PML recruited TET2, regulated DNA modification, reactivated methylation-silenced genes, and impaired cell proliferation. Knockout of PML abolished doxorubicin-promoted DNA modification. In addition, PML and TET2 levels positively correlated with improved overall survival in patients with head and neck cancer. These findings shed insight into the regulatory mechanisms of DNA modification in response to chemotherapeutic agents.Significance: Promyeloctic leukemia protein recruits TET2, regulating DNA modification and cell proliferation in response to chemotherapeutic agents. Cancer Res; 78(10); 2475-89. ©2018 AACR.

Impaired fear extinction in serotonin transporter knockout rats is associated with increased 5-hydroxymethylcytosine in the amygdala.

AIMS: One potential risk factor for posttraumatic stress disorder (PTSD) involves the low activity (short; s) allelic variant of the serotonin transporter-linked polymorphic region (5-HTTLPR), possibly due to reduced prefrontal control over the amygdala. Evidence shows that DNA methylation/demethylation is crucial for fear extinction in these brain areas and is associated with neuronal activation marker c-Fos expression. We hypothesized that impaired fear extinction in serotonin transporter knockout (5-HTT-/- ) rats is related to changes in DNA (de) methylation and c-Fos expression in the prefrontal cortex (PFC) and/or amygdala. METHODS: 5-HTT-/- and 5-HTT+/+ rats were subjected to fear extinction. 2 hours after the extinction session, the overall levels of DNA methylation (5-mC), demethylation (5-hmC), and c-Fos in fear extinction and nonfear extinction rats were measured by immunohistochemistry. RESULTS: 5-HTT-/- rats displayed decreased fear extinction. This was associated with reduced c-Fos activity in the infralimbic PFC. In the central nucleus of the amygdala, c-Fos immunoreactivity was increased in the fear extinction group compared to the no-fear extinction group, regardless of genotype. 5-hmC levels were unaltered in the PFC, but reduced in the amygdala of nonextinction 5-HTT-/- rats compared to nonextinction wild-type rats, which caught up to wild-type levels during fear extinction. 5-mC levels were stable in central amygdala in both wild-type and 5-HTT-/- extinction rats. Finally, c-Fos and 5-mC levels were correlated with the prelimbic PFC, but not amygdala. CONCLUSIONS: In the amygdala, DNA demethylation, independent from c-Fos activation, may contribute to individual differences in risk for PTSD, as conferred by the 5-HTTLPR s-allele.

Immunohistochemical loss of 5-hydroxymethylcytosine expression in acute myeloid leukaemia: relationship to somatic gene mutations affecting epigenetic pathways.

AIMS: Genes affecting epigenetic pathways are frequently mutated in myeloid malignancies, including acute myeloid leukaemia (AML). The genes encoding TET2, IDH1 and IDH2 are among the most commonly mutated genes, and cause defective conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC), impairing demethylation of DNA, and presumably serving as driver mutations in leukaemogenesis. The aim of this study was to correlate 5hmC immunohistochemical loss with the mutation status of genes involved in epigenetic pathways in AML. METHODS AND RESULTS: Immunohistochemical staining with an anti-5hmC antibody was performed on 41 decalcified, formalin-fixed paraffin-embedded (FFPE) bone marrow biopsies from patients with AML. Archived DNA was subjected to next-generation sequencing for analysis of a panel of genes, including TET2, IDH1, IDH2, WT1 and DNMT3A. TET2, IDH1, IDH2, WT1 and DNMT3A mutations were found in 46% (19/41) of the cases. Ten of 15 cases (67%) with TET2, IDH1, IDH2 or WT1 mutations showed deficient 5hmC staining, whereas nine of 26 cases (35%) without a mutation in these genes showed loss of 5hmC. It is of note that all four cases with TET2 mutations showed deficient 5hmC staining. CONCLUSIONS: Overall, somatic mutations in TET2, IDH1, IDH2, WT1 and DNMT3A were common in our cohort of AML cases. Immunohistochemical staining for 5hmC was lost in the majority of cases harbouring mutations in these genes, reflecting the proposed relationship between dysfunctional epigenetic pathways and leukaemogenesis.

Decrease of TET2 expression and increase of 5-hmC levels in myeloid sarcomas.

BACKGROUND: Myeloid sarcoma is a tumor mass that consists of myeloblasts or immature myeloid cells at an extramedullary site. Pathological diagnosis is very difficult based on morphology if systemic signs of disease are absent. The subtype of myeloid sarcoma is also minimally identifiable in the histological picture. FINDINGS: We investigated 18 paraffin-embedded myeloid sarcoma samples, and our immunohistochemical data confirmed the relevance of some key markers for the diagnosis and subclassification of myeloid sarcoma. CD34 was found as a marker in 67% of the myeloid sarcoma cases, and CD34 was positive in all immature types of myeloid sarcoma. CD68 was found in 83% of the myeloid sarcoma cases, but CD68 was most identified in the differentiated type of myeloid sarcoma. Myeloperoxidase (MPO) was positive in all myeloid sarcomas. Notably, the reactivity of MPO in the blastic subtype was much lower in myeloid sarcomas. CD117 reactivity was found in 67% of myeloid sarcomas. Ten-eleven translocation 2 (TET2) protein exhibited significant negative reactivity in 88% of the cases, and 5-methylcytosine (5-hmC) was significantly positive in the nucleus in 100% of the cases. CONCLUSIONS: Our findings indicated that an immunohistochemical panel that included MPO, CD68 and CD34 could be used for the detection of blastic, differentiated and immature types of myeloid sarcoma. Changes in novel epigenetic regulators, including the loss of TET2 and gain of 5-hmC, as characteristics of myeloid malignancies may be useful novel markers of myeloid sarcoma.

Effects of Tet-mediated oxidation products of 5-methylcytosine on DNA transcription in vitro and in mammalian cells.

5-methylcytosine (5-mC) is a well-characterized epigenetic regulator in mammals. Recent studies showed that Ten-eleven translocation (Tet) proteins can catalyze the stepwise oxidation of 5-mC to produce 5-hydroxymethylcytosine (5-HmC), 5-formylcytosine (5-FoC) and 5-carboxylcytosine (5-CaC). The exciting discovery of these novel cytosine modifications has stimulated substantial research interests about their roles in epigenetic regulation. Here we systematically examined the effects of the oxidized 5-mC derivatives on the efficiency and fidelity of DNA transcription using a recently developed competitive transcription and adduct bypass assay. Our results showed that, when located on the transcribed strand, 5-FoC and 5-CaC exhibited marginal mutagenic and modest inhibitory effects on DNA transcription mediated by single-subunit T7 RNA polymerase or multi-subunit human RNA polymerase II in vitro and in human cells. 5-HmC displayed relatively milder blocking effects on transcription, and no mutant transcript could be detectable for 5-HmC in vitro or in cells. The lack of considerable mutagenic effects of the oxidized 5-mC derivatives on transcription was in agreement with their functions in epigenetic regulation. The modest blocking effects on transcription suggested that 5-FoC and 5-CaC may function in transcriptional regulation. These findings provided new evidence for the potential functional interplay between cytosine methylation status and transcription.

Evaluation of global DNA hypomethylation in human prostate cancer and prostatic intraepithelial neoplasm tissues by immunohistochemistry.

OBJECTIVE: To identify the differences of 5-methylcytosine (5-MC) level between primary prostate cancer tissues (PCTs), prostate cancer-adjacent benign tissues (PCABTs), low-grade prostatic intraepithelial neoplasia (LGPIN), and high-grade prostatic intraepithelial neoplasia (HGPIN), and further analysis the 5-MC alterations in prostate cancer with pathologic grade and clinical prognosis. MATERIALS AND METHODS: Immunohistochemistry method with a 5-MC monoclonal antibody was used to identify the 5-methylcytosine (5-MC) levels in PCTs, PCABTs, LGPIN, and HGPIN specimens in the present study. Statistical analysis with SPSS software (SPSS Inc., Chicago, IL) was used to compare differences of 5-MC levels in the four groups and evaluate the 5-MC alterations in prostate cancer with pathologic grade and clinical prognosis. RESULTS: We found that 38 of 48 (79.1%) patients studied showed a decrease in 5-MC staining of PCTs compared with PCABTs. The difference in the methylation levels for the PCTs and the PCABTs was highly statistically significant (P < 0.001). Spearman correlation showed there was no statistically significant association between the average score of 5-MC staining and Gleason score sum. Kaplan-Meier survival analysis showed that patient group with no or weak 5-MC staining compared with group with moderate and strong 5-MC staining was associated with better survival of patients, although there was no statistically significant difference between the 2 groups in predicating prognosis (P = 0.385). The average scores of 5-MC staining for LGPIN, HGPIN, PCABTs, and PCTs groups were 6.91, 1.58, 6.63, and 3.10, respectively. The methylation level of HGPIN group, as well as that of PCTs group, was significantly lower than those of LGPIN (P < 0.001; P < 0.001) and PCABTs groups (P < 0.001; P < 0.001), respectively, with the 5-MC levels of PCABTS group similar to that of LGPIN group (P = 0.476). 5-MC levels of HGPIN group was lower than that PCTs group (P = 0.004). CONCLUSIONS: We found that global DNA methylation was low in most prostate cancer compared with benign regions from the same patient's sections. None of the DNA hypomethylation changes in primary cancers were associated with pathologic grade and clinical prognosis. In addition, immunohistochemistry showed that the global methylation was lower in HGPIN compared with LGPIN and methylcytosine staining in HGPIN was lower than that of PCTs. The results suggest that global DNA hypomethylation might play an important role in the process of prostate cancer initiation rather than progression.

[Cytosine methylation in DNA and its role in cancer therapy]. / Metylacja cytozyny w DNA i jej znaczenie w terapii przeciwnowotworowej.

DNA methyltransferase 1 (DNMT1) is one of three enzymes independently coded in mammalian cells. It catalyses postreplicative synthesis of 5-methylcytosine in DNA. The aim of this modification is regulation of gene expression characteristic for the given organism. DNMT1 maintains methylation pattern by copying it from maternal to daughter stand. S-adenosylo-L-methionina is a donor of methyl group in his process. Disturbance in methylation level results in changes in cell differentiation and finally to tumor transformation. Hypermethylation of promotor or first exon of tumor suppression gene results in silencing of its transcription. While hypomethylation of regulatory sequence of protooncogene and retrotransposon make them transcriptionally active. DNMT1 as a key enzyme in maintaing of proper methylation pattern is a attractive target in anti-tumor therapy.

Quantification of leukocyte genomic 5-methylcytosine levels reveals epigenetic plasticity in healthy adult cloned cattle.

Successful somatic cell nuclear transfer (SCNT) requires epigenetic reprogramming of a differentiated donor cell nucleus. Incorrect reprogramming of epigenetic markings such as DNA methylation is associated with compromised prenatal development and postnatal abnormalities. Clones that survive into adulthood, in contrast, are assumed to possess a normalized epigenome corresponding to their normal phenotype. To address this point, we used capillary electrophoresis to measure 5-methylcytosine (5mC) levels in leukocyte DNA of 38 healthy female bovine clones that represented five genotypes from the Simmental breed and four genotypes from the Holstein breed. The estimated variance in 5mC level within clone genotypes of both breeds [0.104, 95% confidence interval (CI): 0.070-0.168] was higher than between clone genotypes (0, CI: 0-0.047). We quantified the contribution of SCNT to this unexpected variability by comparing the 19 Simmental clones with 12 female Simmental monozygotic twin pairs of similar age. In Simmental clones, the estimated variability within genotype (0.0636, CI: 0.0358-0.127) was clearly higher than in twin pairs (0.0091, CI: 0.0047-0.0229). In clones, variability within genotype (0.0636) was again higher than between genotypes (0, CI: 0-0.077). Twins, in contrast, showed lower variability within genotypes (0.0091) than between genotypes (0.0136, CI: 0.00250-0.0428). Importantly, the absolute deviations of 5mC values of individual SCNT clones from their genotype means were fivefold increased in comparison to twins. Further comparisons with noncloned controls revealed DNA hypermethylation in most of the clones. The clone-specific variability in DNA methylation and DNA hypermethylation clearly show that healthy adult SCNT clones must be considered as epigenome variants.

Epigenetic DNA-(cytosine-5-carbon) modifications: 5-aza-2'-deoxycytidine and DNA-demethylation.

DNA (cytosine-5-carbon) methylation is one of the hallmarks of mammalian chromatin modifications. Distinct methylation pattern can generate synergistic or antagonistic interaction affinities for CpG-islands associated with methylated or unmethylated cytosine binding proteins, which also may dictate histone modifications and dynamic transition between transcriptionally silent or transcriptionally active chromatin states. The enzymes and cofactors associated with DNA-methylation reactions are convincing in terms of chemistry and chemical thermodynamics. The mechanism of demethylation, the candidate enzyme(s) exhibiting direct demethylase activity, and associated cofactors are not firmly established. Use of azanucleosides, such as 5-azacytidine and 5-aza-2'-deoxycytidine (AzadC), in cell culture produces re-expression of certain genes, which otherwise were repressed in association with hypermethylated CpG-rich promoters. Hence the notion developed that AzadC is a demethylating agent. Here we discuss the broad global pictures with the following points: first, chemical definition and recent advances regarding the mechanism of DNA (cytosine-5-carbon) methylation ((Me)CpG-DNA or (Me)CpNpG-DNA formation) and (Me)CpG/(Me)CpNpG-DNA-demethylation, and then with the mechanistic basis of inactivation of DNA-methyltransferase 1 by AzadC. This will clarify that: (i) AzadC has nothing to do with DNA-demethylation; (ii) it cannot prevent even de novo methylation in non-replicating cells; (iii) it can only prevent replication coupled maintenance as well as de novo methylations. Finally, we would like to suggest that terming/designating AzadC as DNA-demethylating agent is a serious misuse of chemistry and chemical terminology.